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学科主题: 分析化学
题名: 电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白肽链和辅基的相互作用
其他题名: Studies on the interaction of polypeptide of cytochrome Tb5 and its F35Y mutant with heme b by electrospray ionization mass spectrometry
作者: 余翀天 ; 郭寅龙 ; 王韵华 ; 姚萍 ; 黄仲贤 ; 张尊建 ; 相秉仁
通讯作者: 郭寅龙
刊名: 化学学报
发表日期: 2001-01-01
卷: 59, 期:6, 页:956-960
收录类别: SCI
部门归属: 中国科学院上海有机化学研究所分析化学实验室; 复旦大学; 中国药科大学分析计算中心
摘要: 本文通过牛肝线粒体细胞色素Tb5和它的F35Y突变体蛋白相对分子质量外标法测, 得到细胞色素Tb5 全蛋白的相对分子质量为10077.5;脱辅基蛋白的相对分子质量为9461.4.F35Y突变体全蛋白的相对分子质量为 10093.6,它的脱辅基蛋白的相对分子质量为9477.5.不同nozzle电压下的电喷雾质谱结果表明,该电压的大小明显影响蛋白肽链与血红素辅基之间的非共价结合.随着电压的降低,全蛋白质谱峰的强度逐渐增大.然而,过低的电压导致了Na+,K+ 离子加合峰相对强度的增加,而不利于谱图分析.同时,考虑到细胞色素Tb5在甲醇溶液和酸性溶液中的变性行为,因此选择nozzle电压70V,10%的甲醇水溶液和 pH=7为得到全蛋白质谱峰的最佳条件.相同实验条件下得到的野生型 Cyt Tb5和F35Y突变体全蛋白的质谱峰相比较,其相对丰度有悬殊的差异,表明F35Y突变体蛋白的血红素结合能力明显低于野生型蛋白.通过对解离出的 Heme b 的分子离子峰进行解析,证明铁仍以三价离子存在于血红素辅基中.
英文摘要: Cytochrome Tb5 from bovine liver mitochondria and its F35Y mutant, digested from cyt b5 membrane protein by trypsin, is a kind of electron transfer protein, which consists of 82 amino acid residues. The molecular weight of the F35Y mutant, 9,4775, is obtained by ESI/TOFMS in extemal standardcalibration method. Compared with molecular weight of the cyt Tb5, 9 461, this data is exactly coincided with the calculated value according to the mutant amino acid sequence. For studtying the stability of heme binding to the F35Y mutant and cyt Tb5, a seres of experimental conditions were selected such as nozzle potetial, organic solvent, pH value, etc. It was found that the aboundance of holoprotein content measured in the mass spectrum decreased with increase of the nozzle potential or / and increase of the composition of organic solvent or/and pH value deviated from neutral. Relatively, the holoprotein's mass abundance of cyt Tb5 is higher than that of F35Y mutant that indicates stronger binding of henme b to polypeptide chain in the wild type cyt Tb5. However, we found that if the nozzle potential was too low it would produce more metal plus ion peaks,suppressing the sample's signals of mass spectrum, and meanwhile increasing organic solvent would reduce stability of the holoprotein. So, the optimal conditions chosen are those; the composition of methanol is 10% and the nozzle potetial is 70 V. In addition, it is verified that the heme b dissociatedfrom the F35Y mutant is a Fe(III) porphyrin by means of internal standard method.
语种: 中文
内容类型: 期刊论文
URI标识: http://ir.sioc.ac.cn/handle/331003/19806
Appears in Collections:分析化学研究室_期刊论文

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Recommended Citation:
余翀天,郭寅龙,王韵华,等. 电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白肽链和辅基的相互作用[J]. 化学学报,2001,59(6):956-960.
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